Aural cholesteatomas arise from the tympanic membrane as a sequela of otitis media. Cholesteatomas are progressive, epithelial lesions that destroy the bony structures of the middle and, sometimes, inner ear. Once established, cholesteatomas can only be mandated by surgical eradication and secondary middle ear reconstruction. The long-term objective of the Principal Investigator's research program is to understand the cellular mechanisms that lead to the development, progression, and bone destruction of cholesteatomas. Such an under- standing may lead to strategies for the non-surgical prevention and management of this disease. The present application is designed to investigate the relationship of two important intercellular signaling molecules, interleukin-1 (IL-1) and nitric oxide (NO), to osteoclastic bone erosion in cholesteatoma. The specific aims of this application are: 1) to determine the expression of the interleukin family of cytokines (IL-1 alpha, IL-1 beta, and IL-1ra) in experimental cholesteatoma and also during osteoclast recruitment and activation induced by pressure in the gerbil bulla model of synchronous bone modeling; and 2) to investigate isoforms of nitric oxide synthase (NOS I, II, III) in the local control of osteoclasts in vitro and in vivo. There are two broad hypothesis, each focused on the isoforms of IL-1 and NOS. For Specific Aim1, hypothesis 1 will be tested: IL-1 isoforms are transcribed within bone lining cells and fibroblasts of the sub- epithelium subjacent to cholesteatoma and in the pressurized bulla and represent an early signal for the recruitment and activation of osteoclasts at a local site. Experimental gerbilline cholesteatoma, gerbilline pressurized bulla, PCS, cloning of PCR products, DNA sequencing, quantitative RT-PCR, bone histomorphometry, and RNA in situ hybridization will be used to test this first hypothesis. For Specific Aim 2, hypothesis 2 will be tested: NO is produced by up- regulation of NOS isoforms within bone lining cells and fibroblasts of the sub-epithelium subjacent to cholesteatoma and in the pressurized bulla. This leads to the local elaboration of NO and the recruitment and activation of osteoclasts. Mouse calvarial organ culture (elemental calcium release and 45Ca release), gerbilline pressurized bulla, PCR, cloning of PCR products, DNA sequencing, quantitative RT- PCR, bone histomorphometry, RNA in situ hybridization, and NADPH- diaphorase histochemistry will be used to test this second hypothesis.